Registration Dossier

Administrative data

Description of key information

Skin irritation (OECD 439 and 431): not irritating

Eye irritation (OECD 492): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 - 27 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted in 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EU method B.40 BIS. (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
adopted in 2008
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Source strain:
other: Keratinocyte strain: 00267
Justification for test system used:
The EpiDerm™ tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™, EPI-200-SCT
- Tissue batch number(s): 25832
- Delivery date: 25 July 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The inserts were thoroughly rinsed with DPBS, blotted with sterile cellulose tissue and set into the respective holding plate, using the wells containing assay medium.
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Anthos Reader 2010 Flexi, Anthos Microsysteme GmbH
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.466 ± 0.03 (acceptance criteria: OD (540 - 570 nm) = 1.0 - 3.0)
- Barrier function: 6.06 h (acceptance criteria: ET 50 = 4.77 - 8.72 h)
- Morphology: viable basal cell layer, intermediate spinous and granular layers and a functional stratum corneum
- Contamination: no
- Reproducibility: yes

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
And if criteria was met for corrosiveness and if the viability after 3 minutes exposure was less than 25%, then the substance is classified as Skin Corrosive Subcategory 1A, while a viability after 3 minutes exposure greater or equal to 25% would lead to a classification as Skin Corrosive Subcategory 1B or 1C
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 μL (a nylon mesh was added in order to ensure sufficient contact with the tissue surface (tissue size is not reported)
- Concentration: undiluted

NEGATIVE CONTROL
- Amount(s) applied: 50 μL
- Concentration: undiluted

POSITIVE CONTROL
- Amount(s) applied: 50 μL
- Concentration: 8M
Duration of treatment / exposure:
3 min / 60 min
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value of 2 replicates, test substance, 3 min exposure
Value:
96.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value of 2 replicates, test substance, 60 min exposure
Value:
91.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value of 2 replicates, positive control, 3 min exposure
Value:
27.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value of 2 replicates, positive control, 60 min exposure
Value:
8.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: tested, but no reduction was observed
- Colour interference with MTT: tested, but no interference was observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: not in the report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (OD = 1.7 (3 min and 1 h) ≥ 0.8 and ≤ 2.8 and within historical control range (3 min: 1.197 - 3.077 and 60 min: 1.377 - 2.571))
- Acceptance criteria met for positive control: yes (viabilty at 3 min = 27.9% < 50% and at 1 h = 8.1% < 15% and within historical control range (3 min: 9.6 - 57.3% and 60 min: 4.1 - 24.2%))
- Acceptance criteria met for variability between replicate measurements: yes, regarding to OECD TG 431 criteria (In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%

Table 1: MTT assay after 3 min exposure

Negative control

Positive control

Test item

Tissue sample

1

2

1

2

1

2

OD570

1.681

1.749

0.503

0.522

1.674

1.661

1.719

1.729

0.498

0.518

1.676

1.663

1.729

1.728

0.493

0.520

1.671

1.677

OD570(mean-blank)

1.671

1.697

0.459

0.481

1.635

1.628

OD570(mean values)

1.684

0.470

1.632

rel. viability (%)

100.0

27.9

96.9

rel. SD

1.1

3.3

0.3

Table 2: MTT assays after 60 min exposure

Negative control

Positive control

Test item

Tissue sample

1

2

1

2

1

2

OD570

1.710

1.721

0.170

0.167

1.532

1.584

1.715

1.675

0.171

0.180

1.542

1.600

1.709

1.702

0.172

0.181

1.537

1.614

OD570(mean-blank)

1.673

1.661

0.132

0.137

1.498

1.561

OD570(mean values)

1.667

0.135

1.530

rel. viability (%)

100.0

8.1

91.8

rel. SD

0.5

2.6

2.9

Interpretation of results:
other: not corrosive (Skin Irrit. 2 or not classified according to Regulation (EC) No 1272/2008)
Conclusions:
Under the conditions of the test, the test substance was shown to have no corrosive potential towards reconstructed human epidermis tissue in the EpiDerm™ model. The result does not allow for the non-classification or classification as irritant of the test substance and therefore further evaluation and/or data generation is required.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Aug - 15 Sep 17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted in 2015
Deviations:
yes
Remarks:
after the post-treatment incubation: additional medium exchange and transfer of the membranes into new wells and 16 h of incubation
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
adopted in 2012
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Source strain:
other: Keratinocyte Strain: 00267
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™-Kit, EPI-200-SIT
- Tissue batch number(s): 25841

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C, 5.0 ± 0.5% CO2
- Temperature of post-treatment incubation: 37 ± 1°C, 5.0 ± 0.5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissue was rinsed in 1-min intervals; After rinsing, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 180 min
- Spectrophotometer: spectrophotometer
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.27 ± 0.071
- Barrier function: 6.28 h
- Morphology: viable basal cell layer, intermediate spinous and granular layers and a functional stratum corneum
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive or irritant to skin if the mean relative viability after 60 minutes exposure is less or equal to 50% of the negative control.
- The test substance is considered to be non-corrosive or non irritant to skin if the viability after 60 minutes exposure is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 30 μL (a nylon mesh was added in order to ensure sufficient contact with the tissue surface (0.63 cm²))
- Concentration: undiluted

NEGATIVE CONTROL
- Amount(s) applied: 30 µL
- Concentration: undiluted

POSITIVE CONTROL
- Amount(s) applied: 30 µL
- Concentration: 5%
Duration of treatment / exposure:
60 min (35 min at 37°C)
Duration of post-treatment incubation (if applicable):
22 h (after transfer to new 6 well-plates containing fresh medium), 16 h (after another transfer into the lower row of the 6-well-plate; no justification for this extra step)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value of 3 replicates, test substance
Value:
93.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value of 3 replicates, positive control
Value:
2.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: not in the report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (mean OD = 2.0)
- Acceptance criteria met for positive control: yes (mean viability = 2.5%)
- Acceptance criteria met for variability between replicate measurements: yes (0.2 - 5.2%)

Table 1: Results MTT assay after 3 h exposure

Substance

Tissue No.

OD570

OD570(mean per tissue - blank)

OD570(mean of 3 tissues)

Cell viability [%]

Cell viability (mean) [%]

Standard Deviation of cell viability [%]

Negative Control

1

2.080

2.043

1.965

-

100.0

-

2.104

2

2.073

2.003

2.031

3

1.892

1.849

1.903

Positive Control

1

0.102

1.824

1.832

2.7

2.5

0.2

0.101

2

0.095

1.811

2.3

0.094

3

0.096

1.861

2.3

0.094

Test substance

1

1.897

0.053

0.048

92.8

93.2

1.3

1.849

2

1.869

0.046

92.2

1.851

3

1.922

0.046

94.7

1.897

 

Interpretation of results:
other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 - 31 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
adopted in 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
human
Strain:
other: EpiOcular™, OCL-200-EIT,
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
The EpiOcular™ Eye Irritation Test (EIT) predicts the acute ocular irritation potential of chemicals by measurement of its irreversible tissue damage caused by cytotoxic effects in the human cornea model. Within a testing strategy, the EpiOcular™ EIT is used as a replacement of the Draize Eye Irritation Test. It is utilized for the classification and labelling of chemicals concerning their eye irritation potential. The EpiOcular™ EIT is intended to differentiate substances that are “not eye irritant” from those that require labelling as either GHS category 1 or 2 for serious eye damage resp. eye irritation potential.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The EpiOcular™ tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcular™ tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm². Analyses for tissue functionality and for potential contaminants was passed.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): undiluted
Duration of treatment / exposure:
28 min
Duration of post- treatment incubation (in vitro):
105 min
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used :
EpiOcular™ tissue model was used. After the assessment and exclusion of direct MTT reduction and colouring potential of the test substance, the well plates were prepared and the tissues were pre-incubated in warm medium under standard culture conditions (37 ± 1 °C, 5 ± 1% CO2 and 80 - 100% relative humidity). After overnight incubation, the tissues were pre-wetted with 20 μL DPBS buffer and then incubated at standard culture conditions for 30 min. Afterwards, 50 μL of the controls and the neat test substance were applied and incubated for 28 min at 37.0 ± 1.0 °C. After exposure the tissue constructs were thoroughly rinsed with DPBS and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 105 min at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment. Therefore, a 24-well-plate was prepared with freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 180 min at standard culture conditions. At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing isopropanol, taking care that no isopropanol is flowing into the tissue insert. The plate was sealed and stored in the refrigerator overnight. On the next day, the plate was shaken for 2 h at room temperature, protected from light. The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μL solution (each) were pipetted into a 96-wellplate. Eight wells with 200 μL isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm.

- RhCE tissue construct used, including batch number :
Keratinocyte strain: 4F1188, Lot Number: 27002

- Doses of test chemical and control substances used :
50 μL of undiluted test substance, 50 μL of demineralised water (negative control), 50 μL of methyl acetate (positive control)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
28 min at 37.0 ± 1.0 °C, 12 min immersion incubation at room temperature, 105 min at 37 ± 1.0 °C post-exposure incubation

- Number of tissue replicates used per test chemical and controls (positive control, negative control) : 2

- Wavelength used for quantifying MTT formazan : 570 nm

- Description of the method used to quantify MTT formazan : The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the Anthos Reader 2010 Flexi microtiter plate photometer. Then the mean OD of the blank isopropanol (ODBlk) was calculated, followed by the subtraction of mean ODBlk of each value of the same experiment (corrected values).
The mean OD of the two replicates for each tissue the mean OD of the two relating tissues for controls and test substance were then calculated.

To calculate the relative absorbance, the following equation was used: % viability = (OD corrected of test substance or positive control/ OD corrected of mean negative control)*100

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model :

The in vitro eye irritation test is considered valid if it meets the following criteria:
a) mean OD570 of the negative control: > 0.8 and < 2.5
b) mean relative tissue viability of the positive control: < 50% relative to the negative control.
c) variation within replicates: < 20%
d) values for negative control and for positive control within range of historical data

A test item is considered to be positive in the in vitro eye irritation test if the relative mean tissue viability of two individual tissues after exposure to the test item is ≤ 60% of the mean viability of the negative controls, thus requiring classification for eye irritation (Cat. 2) or serious eye damage (Cat.1).

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : negative control range: 1.253 - 2.437; positive control range: 12.4 - 57.2%

- Complete supporting information for the specific RhCE tissue construct used :
Tissue viability: 1.269 ± 0.039 (accepted range: 1.1 - 3.0)
Barrier function: ET-50: 24.36 min (accepted range: 12.2 - 37.5)
Irritation parameter:
other: mean viability (%)
Remarks:
out of 2 replicates
Run / experiment:
test substance
Value:
102.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: mean viability (%)
Remarks:
out of 2 replicates
Run / experiment:
positive control
Value:
33.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: not in the report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, OD = 2.1 > 0.8 and < 2.5 (historical range: 1.253 - 2.437)
- Acceptance criteria met for positive control: yes, % viability = 33.2% < 50% (historical range: 12.4 - 57.2%)
- Range of historical values if different from the ones specified in the test guideline: 1 - 3.3% < 20%

Table 1: Results of MTT assay (28 min exposure)

 

Tissue No.

OD570

Mean (OD570) - blank

Mean tissue viability (% of negative control)

Negative

control

1

2.156

2.137

100

2.193

2

2.084

2.067

2.125

Positive control

1

0.750

0.721

33.9

33.2

0.750

2

0.716

0.685

32.6

0.730

Test substance

1

2.174

2.143

102.0

102.4

2.188

2

2.184

2.164

102.9

2.219

  OD = optical density

Interpretation of results:
other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

The skin corrosion potential of the test substance was assessed in an in vitro skin corrosion test using Reconstructed Human Epidermis (RHE) tissue according to OECD guideline 431 and in compliance with GLP (Evonik Dr Straetmans, 2017). The EpiDermTM(EPI-200-SCT) model was used, with 2 replicate tissues. After treatment with the test substance the tissue viability did not decrease below the threshold of 50% (3 min. exposure: 96.9%; 1 h exposure: 91.8%) compared with the negative control. The negative and positive controls were valid. Therefore, the test substance is not considered be corrosive towards the skin.

In order to determine the classification or non-classification of the test substance and additional in vitro skin irritation test was performed: the in vitro skin irritation test using Reconstructed Human Epidermis (RHE) tissue according to OECD guideline 439 and in compliance with GLP (Evonik Dr Straetmans, 2017). The EpiDermTM(EPI-200-SIT) model was used, with 3 replicate tissues. After treatment with the test substance for 60 min. the tissue viability was 93.2%, compared with the negative control. The negative and positive controls were valid. The test substance is considered to possess no irritant potential. In conclusion, based on available studies, the test substance is considered not to be irritating or corrosive towards the skin.

 

Eye irritation

The eye irritation potential of the test substance was assessed in an in vitro eye irritation test using a Reconstructed Human Cornea-like Epithelium (RhCE) model according to OECD guideline 492 and in compliance with GLP (Evonik Dr Straetmans, 2017). The EpiOcularTMtissue model was used, with 2 replicate tissues per test. The negative and positive controls were valid. After treatment with the test substance the tissue viability was measured to be 102.4% compared with the negative control. Therefore, the test substance is not considered be irritant towards the eye.

Justification for classification or non-classification

The available data on skin and eye irritation do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.